The functional association between the sodium/bicarbonate cotransporter (NBC) and the soluble adenylyl cyclase (sAC) modulates cardiac contractility.

The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na+/HCO3- cotransporter (NBC) promotes the cellular co-influx of HCO3- and Na+. Since sAC activity is regulated by HCO3-, our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca2+ transient (Ca2+i) by epifluorescence, Ca2+ sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pHi) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 µM), the selective inhibitor of sAC KH7 (1 µM), and the PKA inhibitor H89 (0.1 µM) induced a negative inotropic effect which was associated with a decrease in Ca2+i. Since PKA increases Ca2+ release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO3- influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca2+ handling and basal cardiac contractility.

The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30).

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na+ and HCO3- into the cytoplasm upon intracellular acidosis, restoring the acidic pHi to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na+:1HCO3-) and the electrogenic NBCe1 (1Na+:2HCO3-). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.

Aldosterone stimulates the cardiac sodium/bicarbonate cotransporter via activation of the g protein-coupled receptor gpr30.

Some cardiac non-genomic effects of aldosterone (Ald) are reported to be mediated through activation of the classic mineralocorticoid receptor (MR). However, in the last years, it was proposed that activation of the novel G protein-coupled receptor GPR30 mediates certain non-genomic effects of Ald. The aim of this study was to elucidate if the sodium/bicarbonate cotransporter (NBC) is stimulated by Ald and if the activation of GPR30 mediates this effect. NBC activity was evaluated in rat cardiomyocytes perfused with HCO3(-)/CO2 solution in the continuous presence of HOE642 (sodium/hydrogen exchanger blocker) during recovery from acidosis using intracellular fluorescence measurements. Ald enhanced NBC activity (% of ΔJHCO3(-); control: 100±5.82%, n=7 vs Ald: 151.88±11.02%, n=5; P<0.05), which was prevented by G15 (GPR30 blocker, 90.53±7.81%, n=7). Further evidence for the involvement of GPR30 was provided by G1 (GPR30 agonist), which stimulated NBC (185.13±18.28%, n=6; P<0.05) and this effect was abrogated by G15 (124.19±10.96%, n=5). Ald- and G1-induced NBC stimulation was abolished by the reactive oxygen species (ROS) scavenger MPG and by the NADPH oxidase inhibitor apocynin. In addition, G15 prevented Ald- and G1-induced ROS production. Pre-incubation of myocytes with wortmannin (PI3K-AKT pathway blocker) prevented Ald- or G1-induced NBC stimulation. In summary, Ald stimulates NBC by GPR30 activation, ROS production and AKT stimulation.

Characterization of the Na/HCO3(-) cotransport in human neutrophils.

BACKGROUND: Bicarbonate transport has crucial roles in regulating intracellular pH (pHi) in a variety of cells. The purpose of this study was to evaluate its participation in the regulation of pHi in resting and stimulated human neutrophils. METHODS: Freshly isolated human neutrophils acidified by an ammonium prepulse were used in this study. RESULTS: We demonstrated that resting neutrophils have a bicarbonate transport mechanism that prevents acidification when the Na(+)/H(+) exchanger is blocked by EIPA. Neutrophils acidified by an ammonium prepulse showed an EIPA-resistant recovery of pHi that was inhibited by the blocker of the anionic transporters SITS or the Na(+)/HCO3(-) cotransporter (NBC) selective inhibitor S0859, and abolished when sodium was removed from the extracellular medium. In western blot and RT-PCR analysis the expression of NBCe2 but not NBCe1 or NBCn1 was detected in neutrophils Acidified neutrophils increased the EIPA-insensitive pHi recovery rate when its activity was stimulated with fMLF/ cytochalasin B. This increase in the removal of acid equivalents was insensitive to the blockade of the NADPH oxidase with DPI. CONCLUSION: It is concluded that neutrophils have an NBC that regulates basal pHi and is modulated by chemotactic agents.

Reduced sarcolemmal expression and function of the NBCe1 isoform of the Na⁺-HCO3⁻ cotransporter in hypertrophied cardiomyocytes of spontaneously hypertensive rats: role of the renin-angiotensin system.

AIMS: Electroneutral (NBCn1) and electrogenic (NBCe1) isoforms of the Na(+)-HCO3(-) cotransporter (NBC) coexist in the heart. We studied the expression and function of these isoforms in hearts of Wistar and spontaneously hypertensive rats (SHR), elucidating the direct implication of the renin-angiotensin system in the NBC regulation. METHODS AND RESULTS: We used myocytes from Wistar, SHR, losartan-treated SHR (Los-SHR), and Angiotensin II (Ang II)-induced cardiac hypertrophy. We found an overexpression of NBCe1 and NBCn1 proteins in SHR that was prevented in Los-SHR. Hyperkalaemic-induced pHi alkalization was used to study selective activation of NBCe1. Despite the increase in NBCe1 expression, its activity was lower in SHR than in Wistar or Los-SHR. Similar results were found in Ang II-induced hypertrophy. A specific inhibitory antibody against NBCe1 allowed the discrimination between NBCe1 and NBCn1 activity. Whereas in SHR most of the pHi recovery was due to NBCn1 stimulation, in Wistar and Los-SHR the activity of both isoforms was equitable, suggesting that the deteriorated cardiac NBCe1 function observed in SHR is compensated by an enhanced activity of NBCn1. Using the biotin method, we observed greater level of internalized NBCe1 protein in SHR than in the non-hypertophic groups, while with immunofluorescence we localized the protein in endosomes near the nucleus only in SHR. CONCLUSIONS: We conclude that Ang II is responsible for the impairment of the NBCe1 in hypertrophied hearts. This is due to retained transporter protein units in early endosomes. Moreover, NBCn1 activity seems to be increased in the hypertrophic myocardium of SHR, compensating impaired function of NBCe1.

Modulation of the cardiac sodium/bicarbonate cotransporter by the renin angiotensin aldosterone system: pathophysiological consequences.

The sodium/bicarbonate cotransporter (NBC) is one of the major alkalinizing mechanisms in the cardiomyocytes. It has been demonstrated the existence of at least two functional isoforms, one that promotes the co-influx of 1 molecule of Na(+) per 1 molecule of HCO(-) 3 (electroneutral isoform; NBCn1) and the other one that generates the co-influx of 1 molecule of Na(+) per 2 molecules of HCO(-) 3 (electrogenic isoform; NBCe1). Both isoforms are important to maintain intracellular pH (pH i ) and sodium concentration ([Na(+)] i ). In addition, NBCe1 generates an anionic repolarizing current that modulates the action potential duration (APD). The renin-angiotensin-aldosterone system (RAAS) is implicated in the modulation of almost all physiological cardiac functions and is also involved in the development and progression of cardiac diseases. It was reported that angiotensin II (Ang II) exhibits an opposite effect on NBC isoforms: it activates NBCn1 and inhibits NBCe1. The activation of NBCn1 leads to an increase in pH i and [Na(+)] i , which indirectly, due to the stimulation of reverse mode of the Na(+)/Ca(2+) exchanger (NCX), conduces to an increase in the intracellular Ca(2+) concentration. On the other hand, the inhibition of NBCe1 generates an APD prolongation, potentially representing a risk of arrhythmias. In the last years, the potentially altered NBC function in pathological scenarios, as cardiac hypertrophy and ischemia-reperfusion, has raised increasing interest among investigators. This review attempts to draw the attention on the relevant regulation of NBC activity by RAAS, since it modulates pH i and [Na(+)] i , which are involved in the development of cardiac hypertrophy, the damage produced by ischemia-reperfusion and the generation of arrhythmic events, suggesting a potential role of NBC in cardiac diseases.